Methods and apparatus using electrostatic atomization to form liquid vesicles

ABSTRACT

The methods of the invention employ electrostatic atomization to form a compound droplet of at least two miscible fluids. The compound droplet comprises a core of a first fluid and a layer of a second fluid completely surrounding the core. The first fluid contains the agent to be encapsulated and the second fluid contains an encapsulating agent. The first and second liquids are miscible. The encapsulated droplets can contain a variety of materials including, but not limited to, polynucleotides such as DNA and RNA, proteins, bioactive agents or drugs, food, pesticides, herbicides, fragrances, antifoulants, dyes, oils, inks, cosmetics, catalysts, detergents, curing agents, flavors, fuels, metals, paints, photographic agents, biocides, pigments, plasticizers, propellants and the like and components thereof. The droplets can be encapsulated by a variety of materials, including, but not limited to, lipid bilayers and polymer shells. An additional complete or partial layer of a third fluid can be formed on the outside of the second fluid layer. The third fluid can contain a targeting or steric stabilizing agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional ApplicationNo.60/470,287, filed May 14, 2003, which is hereby incorporated byreference to the extent not inconsistent with the disclosure herein.

BACKGROUND OF THE INVENTION

This invention is in the field of encapsulation, in particular methodsand apparatus for encapsulation of liquid droplets. The methods of theinvention employ electrostatic atomization to form a compound dropletfrom at least two miscible fluids. The compound droplet comprises a coreof a first fluid and a layer of a second fluid completely surroundingthe core. The first fluid contains an agent to be encapsulated and thesecond fluid contains an encapsulating agent.

Encapsulation is used in a variety of well-known applications such asscratch-and-sniff perfumes, carbonless copy paper, laundry detergent,packaged baking mixes, and pharmaceutical drugs for taste masking andsustained release. Commercial techniques for encapsulation includecomplex coacervation, co-extrusion, interfacial polymerization,desolvation and various coating techniques. Since the 1930s, a number ofvariations to the emulsion encapsulation technique have evolved.Mathiowitz et al (U.S. Pat. No. 6,143,211) describe a process forpreparing microparticles through phase inversion.

Several encapsulation methods involve flow of two liquids throughconcentric orifices to form a droplet within a droplet, which can alsobe called a compound droplet. Merrill et al. (U.S. Pat. No. 2,275,154)describes compound droplets formed by gravity. Raley et al. (U.S. Pat.No. 2,766,478) describe compound droplets formed by a combination ofgravity and a slight pneumatic pressure. Somerville describes compounddroplets formed by a centrifugal encapsulating apparatus (U.S. Pat. No.3,015,128 and U.S. Pat. No. 3,310,612). Somerville also describescompound droplets formed by extruding concentrically arranged fluid rodsof film and filler material into a stream of carrier fluid. The speed ofthe carrier fluid is selected to cause the rod to elongate and break upinto segments which form into individual particles (U.S. Pat. No.3,389,194). Ganna-Calvo describes methods for manufacturing coateddroplets in which a gas focuses two concentrically positioned immisciblestreams to a stable unified jet which flows out of the chamber exitorifice and breaks up into coated particles (U.S. Pat. No. 6,405,936).Methods for forming compound droplets from two immiscible or poorlymiscible fluids have also been described where the droplets are formedby dissociation of stable electrified coaxial jets (WO 02/060591,Loscertales. I.G. et al., Mar. 2002, Science, 295, 1695-1698, and USPatent Application Publication Number 2004/0069632).

Concentric orifice configurations have also been used to formelectrosprays for mass spectrometry applications. Mylchreest et al.describe an electrospray ion source in which the liquid sample issheathed with a sheath liquid (U.S. Pat. No. 5,122,670).

Liposomes encapsulating an aqueous core have been proposed for drugdelivery and gene therapy. The ability of phospholipids to self-assembleinto bilayers enclosing an aqueous core (“liposomes”) was firstdescribed by Bangham almost four decades ago (Bangham et al., 1965). Thedevelopment of liposomes as potential drug delivery vehicles wasintensely studied in the 70s and 80s, and several liposome-basedproducts are currently on the market (Gregoriadis, 1995). Of theseproducts, only three consist of water-soluble drugs that areencapsulated within the lipid envelope. Although liposomes have beenformulated such that a long circulating half-life is achieved, theencapsulation of drugs within the lipid bilayer can be inefficient.Typical encapsulation procedures involve the rehydration of a driedlipid film with a drug-containing solution such that drug isencapsulated upon vesicle formation. This traditional approach yieldsencapsulation efficiencies of <10%, with the bulk of the drug remainingoutside of the liposome (Semple et al., 2001). The removal of theunencapsulated drug is labor-intensive, costly, and results insubstantial losses of both drug and lipid. Encapsulation efficiency canbe improved by utilizing a pH gradient and taking advantage of drugsthat will partition across the membrane and become entrapped within theacidified interior of the liposome (Lasic et al., 1995). Unfortunately,separation of the unencapsulated drug from the loaded liposomes is stillproblematic, and this approach is not applicable to macromoleculartherapeutics that cannot penetrate the bilayer.

One aspect of gene delivery that has been shown to have a major effecton therapeutic gene delivery in vivo is the maintenance of DNA integrityin physiological fluids. More specifically, it has been demonstratedthat the destabilization of non-viral vectors in serum causes theexposure of DNA to nucleases (Li et al., 1999). As a result, DNA israpidly degraded in the blood, thereby preventing it from providing anytherapeutic benefit.

This problem has stimulated interest in identifying lipid formulationsthat bind very strongly to DNA in order to maintain the therapeutic genein a complex that is resistant to nuclease degradation (Li et al.,1999). However, studies have clearly shown that gene expression cannotoccur unless the bound lipid is removed to allow transcription in thenucleus (Zabner et al., 1995; Pollard et al., 1998). Therefore, the useof cationic lipids to prevent DNase degradation can result in a verystable complex that does not disassociate in the intracellularenvironment, and ultimately inhibits therapeutic gene expression(Oupicky et al., 2002). In an effort to circumvent this dilemma, someresearchers have synthesized cationic vectors possessing chemicallinkages that can be degraded in an intracellular environment (Bulmus etal., 2003; Dauty et al., 2001). In this way, the vector remains intactin blood to maintain DNA integrity, but dissociation is aided byenzymatic degradation within the cell to allow gene expression.

The problems with encapsulating negatively-charged macromolecules withintraditional liposomes stimulated Felgner et al. (1987) to utilizecationic liposomes in an effort to improve the efficiency of DNAencapsulation. This landmark study revolutionized gene delivery, andstimulated the use of cationic lipids in synthetic vectors. However,subsequent studies have clearly shown that true encapsulation is rarelyachieved under these conditions, but that an ionic interaction of theDNA with the cationic liposomes causes the formation of a lipid-DNAcomplex that is ultimately responsible for gene delivery.

Another factor that must be considered when administering vectors invivo is the interaction with various components in physiologicalsolutions. For example, it is well known that non-viral vectors bindwith serum proteins upon IV injection (Yang and Huang, 1997; Dash etal., 1999; Faneca et al., 2002; Opanasopit et al., 2002; Trubetskoy etal., 2003). Furthermore, it has been shown that the binding of serumcomponents causes aggregation in vivo, which decreases the circulationhalf-life of the vector (Dash et al., 1999; Oupicky et al., 2002). Somestudies have taken advantage of the vector aggregation to enhance genedelivery to the lung (Li et al., 1999; Barron et al., 1999; Li andHuang, 2000; Liu and Huang, 2002), but safety concerns and the inabilityto target other tissues limit the potential applications of thisapproach. Other researchers have attempted to incorporate high levels ofsteric stabilizers and targeting ligands to prolong circulatinghalf-lives (Choi et al., 1998; Fajac et al., 1999; Ogris et al., 1999;Tam et al., 2000; Oupicky et al., 2002). Although this approach has beensuccessfully utilized for liposome-based pharmaceuticals and appears tobe effective at increasing circulation lifetimes (Papahadjopoulos etal., 1991; Torchilin et al., 1994), studies have also shown that theincorporation of polyethylene glycol (PEG)-conjugated components intovectors disrupts normal cellular processing and ultimately reducestransfection rates (Harvie et al., 2000).

Polymeric vesicles formed from amphiphilic polymers have also beenproposed as drug delivery vehicles. Amphiphilic polymers proposed forpolymeric vesicles include diblock copolymers ofpolyethyleneoxide-polyethylene (Discher, B. M. et al. (1999)“Polymersomes: Tough Vesicles Made from Diblock Copolymers”, Science,284: 1143-1146), carbohydrate-based polymers based on chitosan (Uchegbu,I. F. et al., (1998) “Polymeric chitosan based vesicles for drugdelivery. J. Pharm. Pharmacol., 50, 453-8) and amino acid based polymers(Uchegbu, I. F. et al., (1998) “Polymeric vesicles from amino acidhomopolymers”, Proc. Intl. Symp. Control. Rel. Bioact. Mater. 25,186-7).

SUMMARY OF THE INVENTION

The invention provides methods and apparatus for making encapsulateddroplets. The encapsulated droplets can contain a variety of agents tobe encapsulated including, but not limited to, polynucleotides such asDNA and RNA, chemically modified polynucleotides, polynucleotidecomplexes, proteins, bioactive agents, food, pesticides, herbicides,fragrances, antifoulants, dyes, oils, inks, cosmetics, catalysts,detergents, curing agents, flavors, fuels, metals, paints, photographicagents, biocides, pigments, plasticizers, propellants and the like andcomponents thereof. The droplets can be encapsulated by a variety ofmaterials, including, but not limited to, lipid bilayers and polymershells.

Applications of the encapsulated droplets made by the methods of theinvention include, but are not limited to, polynucleotide delivery. Themethods of the invention can completely encapsulate DNA (either naked orcondensed) within liposomes and potentially circumvent problemsassociated with DNase susceptibility, cationic lipid toxicity,complement activation, serum-induced aggregation, vector dissociation,and “first pass” elimination in the liver and lungs (Devine et al.,1994, Plank et al., 1996; Yew et al., 2001).

Furthermore, the individual assembly of each vector via electrostaticspraying results in a narrow particle size distribution with a lowerlimit on droplet size below 500 nm (note most conventional atomizationtechniques have a 5 to 7 μm limit). Previous studies that producevectors by traditional methods (i.e., the mixing of separate solutionscontaining the various components) have demonstrated that fractionationof the heterogeneous particle preparation yields a small population ofvectors with very high transfection efficiency (Hofland et al., 1996;Gao and Huang, 1996). Therefore, the relatively homogeneous preparationof vectors produced by electrostatic co-extrusion can be prepared withmore uniform physicochemical properties that can be systematicallyoptimized for maximum serum stability and transfection activity.

Efficient encapsulation procedures that allow elimination of cationiclipids from the vector also reduce the interaction with serum componentsand minimize the need for steric stabilization via PEGylation (Nicolazziet al., 2003). Indeed, several liposome-based pharmaceuticals that arecurrently on the market achieve adequate circulation lifetimes withoutsteric stabilization by utilizing neutral and anionic lipids that onlyweakly interact with serum components (e.g., DaunoXome®, Abelcet®,AmBiosome®).

If steric stabilization via PEGylation is employed with the methods ofthe invention, the drawbacks of steric stabilization by PEGylation maybe minimized. In the methods of the invention, the DNA is not physicallybound to the PEGylated lipid, but can be reliably entrapped within theaqueous compartment of a PEGylated liposome. For example, if the lipid“coat” containing the PEG is not electrostatically bound to the DNA,steric stabilization may be achieved in the circulation, but PEGylatedcomponents may be readily dissociated from the transgene to facilitatetransfection.

The electrostatic co-extrusion technique can be applied broadly to anysituation where high encapsulation and consistent particlecharacteristics are desirable. The potential advantages for themanufacture of polymer-encapsulated micro- and nanospheres aresignificant, and certain drugs (e.g., insulin) have an absoluterequirement for a low burst release that is dependent upon completeencapsulation (Yamaguchi et al., 2002). In addition to drug deliveryapplications, the ability to efficiently encapsulate high concentrationsof proteins within a lipid membrane offers the potential to solve manyof the problems associated with the development of artificial blood.More specifically, conventional methods for hemoglobin encapsulation donot permit the high protein concentrations needed to simulate that foundin red blood cells, and the inability to produce consistently small“particle” sizes (200-300 nm) continues to limit the development ofliposome-encapsulated hemoglobin (LEH), despite its distinct advantagesover other non-encapsulated technologies (Riess, 2001).

The process for making the encapsulated droplets uses electrostaticatomization to form a compound droplet. The compound droplet comprises acore of a first fluid and a layer of a second fluid completelysurrounding the core. The compound droplet may comprise one or moreadditional layers completely or partially surrounding the layer ofsecond fluid. The first fluid can either dissolve or suspend the agentto be encapsulated and can be aqueous. The second fluid can eitherdissolve or suspend the encapsulating agent and is selected so that itis miscible with the first fluid. The electrostatic atomization requiresthat at least one of the fluids has sufficient electrical conductivity.

A two-fluid compound droplet is formed as follows. The first and secondfluids are pumped through concentric flow channels such that the firstfluid flows through the inner flow channel. The two fluids exit the flowchannels through concentric orifices. Laminar flow conditions arepreferred, so that the first and second fluids are separated by aninterface upon exiting the orifices. An electrical potential differenceis applied to the apparatus such that that a structured Taylor cone isformed where the second liquid surrounds the first fluid. A coaxial jetis emitted from the Taylor cone. This coaxial jet then breaks up intocompound droplets of a first fluid surrounded by a layer of secondfluid.

After each compound droplet is formed, the encapsulating agent presentin the layer of second liquid forms an encapsulating coating or shell.In an embodiment, the shell forms through at least partial removal ofthe second liquid. In this embodiment, enough of the second liquid isremoved from the liquid environment around the encapsulating agent thatthe encapsulating agent no longer remains in solution. For example,phospholipid encapsulating agents can form a lipid bilayer as the liquidenvironment around the encapsulating agent becomes sufficiently aqueous.The change in liquid environment around the encapsulating agent canoccur as the second liquid evaporates from the droplet, or throughinterdiffusion of the liquids, or through a combination of thesefactors. In a different embodiment, the encapsulating agent forms ashell through cross-linking of the encapsulating agent, e.g. throughphotoirradiation.

In an embodiment, the invention provides electrostatic atomizationapparatus with arrays of nozzles for formation of compound droplets.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates an experimental configuration of theelectrostatic atomization system.

FIG. 2 schematically illustrates a cross-section of the flow channels ofan electrostatic atomization system.

FIG. 3 illustrates the predicted (line) and experimentally observed(dots) variation of methanol droplet diameter with liquid flow rate atatmospheric temperature and pressure.

FIG. 4 illustrated the predicted variation of water droplet diameterwith liquid conductivity at atmospheric temperature and pressure.

FIG. 5. illustrates water droplets (clear interior) in lipid shells(dark wall) formed through electrostatic spraying.

FIG. 6 is a schematic of staggered nozzle array. Filled circlesrepresent the co-axial capillaries producing the compound droplets.Empty circles represent false charged capillaries.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods for making encapsulated droplets throughelectrostatic atomization of at least a first and a second fluid, thefirst fluid comprising the agent to be encapsulated and the second fluidcomprising the encapsulating agent. As used herein, electrostaticatomization is a technique in which electrostatic forces aid in thegeneration of fine liquid droplets. The particular form of electrostaticatomization used in the methods of the invention may also be termedelectrostatic co-extrusion, since it involves co-flowing fluids throughcoaxial flow channels, having coaxial orifices.

In an embodiment, the method comprises the steps of:

-   -   a. forming a compound droplet through electrostatic atomization        of a first and a second liquid, the compound droplet comprising        a droplet of the first liquid surrounded by a layer of the        second liquid, wherein the first liquid comprises an agent to be        encapsulated, the second liquid comprises an encapsulating        agent, the second liquid comprises an encapsulating agent and is        miscible with the first liquid and at least one of the first or        second liquid is electrically conductive; and    -   b. forming an encapsulating shell from the encapsulating agent,        thereby forming an encapsulated droplet comprising an agent to        be encapsulated.

FIG. 1 shows a schematic of an electrostatic atomization apparatus. Themajor components include a flow channel assembly 60. The flow channelassembly 60 comprises two concentric flow channels, a first, inner flowchannel 22 and a second, outer flow channel 24 with concentric lowerorifices 23 and 25. A first pump 12 supplies a controlled flow of theinner fluid 1 through the inner flow channel 22. Similarly, a secondpump 14 delivers a controlled flow of the outer fluid 2 through theouter flow channel 24 in the annular space between the inner (first) andouter (second) flow channels 22, 24. The inner and outer fluids aresimultaneously extruded at the lower orifices of the flow channels 22,24. The lower orifices of the flow channels may be said to form theoutlet of a nozzle. The inner or first fluid contains the agent to beencapsulated, and the outer or second fluid contains the encapsulatingagent. In FIG. 1, the flow channels are shown as capillary tubes ofconstant cross-section. The diameter of the orifices is sufficientlysmall that droplet formation occurs through electrospray rather thangravity driven dripping. The diameter of the flow channels need not beof constant cross-section. For example, the walls of the flow channelsmay converge as they approach the orifice or outlet. Furthermore, theapparatus can be configured with additional coaxial flow channels toproduce compound droplets with additional layers (e.g. a third coaxialflow channel surrounding the second flow channel for formation of athird layer). Electrostatic atomization apparatus suitable for use withthe invention may be operated at atmospheric pressure or under vacuum.

In the embodiment shown in FIG. 1, both the first and second fluid aregiven an electric charge by transferring current from a high voltagepower supply 50 through an electrode (e.g., a metal tube) in contactwith the fluid. The current is drawn into the fluid by using anelectrical ground electrode 40 (e.g., a plate or hoop) placed downstreamof the capillary exits. For a fluid to be given an electric charge inthis manner, the fluid must be sufficiently electrically conductive. Asused herein, an electrically conducting fluid is sufficiently conductivethat the fluid can be given an electrical charge in this manner. Therequired fluid conductivity depends in part on the density of the fluid.In an embodiment, fluid conductivities of greater than about 1×10⁻⁹(cm⁻¹Ohms⁻¹) are sufficiently conductive.

In FIG. 1, the potential applied to both flow channels is the same.However, variable resistors can be used to manipulate the potentialapplied to each flow channel, and thus the current applied to eachfluid. In this embodiment, the fluid streams are electrically insulatedfrom each other while in the flow channels. In other embodiments, onlythe first or the second fluid can be given an electric charge.

In an embodiment, the flow channels are negatively charged.

In one embodiment, the ground plate 40 is placed approximately 10 mmbelow the capillary orifices. The ground plate distance can vary fromabout 1 mm to about 50 mm, depending on the applied voltage, flow rates,fluid properties, and the desired droplet size.

The potential difference between the charged fluid(s) and the groundplate 40 pulls the fluid(s) down, opposing surface tension andaccelerating liquid column breakup. If only one fluid is charged, themotion of that fluid pulls on the fluid in the other flow channel. Forexample, if only the inner or first fluid is charged, the motion of theinner fluid pulls on the fluid in the outer capillary causing it tobecome encased. In an embodiment, the voltage used to drive the processranges between about four and about six thousand volts (depending onflow channel orifice-to-ground distance).

As used herein, one fluid is miscible in another if it is at leastpartially able to mix with or dissolve into the other fluid. The firstand second fluids may be partially or completely miscible with eachother. In an embodiment, the first and second fluids are not poorlymiscible with one another. It has been found that miscible fluids canrequire more care than immiscible systems in establishing well behavedTaylor cones. It is important to maintain laminar flow of misciblefluids as they exit the flow channels. The flow behavior of the fluidsdepends upon the fluid flow rates, with lower flow rates being moreconducive to laminar flow. Stable interfaces are more likely and betterresults will be obtained if the velocity profiles are similar across theexits of all geometries. In an embodiment, the velocity profiles aresubstantially uniform across the exits of all geometries. Laminar flowis also more likely to be obtained if the velocity fields of the innerand outer fluids are similar to one another. In an embodiment, thevelocity fields of the inner and outer fluids are matched. The flowbehavior also depends upon the configuration of the outlets of the flowchannels. Preferably, the outlet of the inner flow channel is flush ordownstream of the outlet of any surrounding flow channels. In anembodiment, the outlet of the inner flow channel projects less thanabout 2 mm from the outlets of any surrounding flow channels. Inaddition, the shape of the flow channel walls can affect the flowbehavior. In an embodiment, the walls of the second flow channelconverge near the outlet of that flow channel, as illustratedschematically in FIG. 2. In another embodiment, the walls of both thefirst and second flow channel converge near their respective outlets.

The droplet size is a function of the liquid properties, flow rate, andapplied charge. Previous research on the formation dynamics andcharacterization of aerosols from electrostatic sprays indicate theexpected versatility in the particle size produced by this techniqueranges from hundreds of microns to tens of nanometers (Gana-Calvo, 1997;Canna-Calvo, 1999; De Juan and de la Mora, 1997; Rosell-Llompart and dela Mora, 1994). The size of simple droplets (i.e. not compound droplets)can be predicted using a universal scaling law where the dropletdiameter (d) is largely a function of flow rate (Q), fluid density (ρ),fluid conductivity (K), and surface tension (γ): $\begin{matrix}{{d = {{\frac{2.9}{\pi^{1/3}}\left\lbrack \frac{ɛ_{o}\rho}{K\quad\gamma} \right\rbrack}^{1/6}Q}}{{where}\quad ɛ_{o}\quad{is}\quad{the}\quad{permittivity}\quad{of}\quad a\quad{vacuum}}} & \left( {{Equation}\quad 1} \right)\end{matrix}$

FIGS. 3 and 4 demonstrate the impact of flow rate and conductivity onthe diameter of simple droplets formed at atmospheric temperature andpressure. Although flow rate appears more than adequate to formparticles well into the submicron range, current experimentalobservations report a lower limit of approximately half a micron indiameter. This occurs because standard syringe pumps operating underextremely low flow rates can produce unsteady, pulsating flows thatnegate several of the assumptions used to produce these theoreticalcurves. As a result, the production of particles smaller than half amicron may require manipulation of other factors in addition to flowrate. Experimentally, it is easy to alter the solution conductivity byadding salts to the solution and thereby reduce droplet size (thepresence of polynucleotides and condensing agents will also increaseconductivity and therefore aid in achieving small particle sizes). FIG.4 demonstrates that particle sizes down to the 100 nm range are feasibleif we account for the conductivity changes arising from moderateincreases in salt or DNA concentration within the aqueous core. Forexample, the conductivity from physiological saline (150 mM NaCl) isapproximately 0.016 S/m resulting in a theoretical particle size of 83nm according to these calculations (FIG. 4).

The compound droplets 30 formed through electrostatic atomizationcomprise a core 32 of a first fluid and a layer 33 of a second fluidcompletely surrounding the core. The compound droplet may comprise oneor more additional layers completely or partially surrounding the layerof second fluid.

After the compound droplets are formed, the encapsulating agent presentin the layer of second liquid forms an encapsulating coating or shell.In an embodiment, the shell forms as the liquid environment around theencapsulating agent becomes sufficiently aqueous that the encapsulatingagent no longer remains in solution. The change in liquid environmentaround the encapsulating agent can occur as the second liquid evaporatesfrom the droplet, as the first and second liquids interdiffuse, orthrough a combination of these factors. In a different embodiment, theencapsulating agent forms a shell through cross-linking of theencapsulating agent, e.g. through photo irradiation.

In order to efficiently encapsulate the agent to be encapsulated,formation of the encapsulating shell should occur before substantialdiffusion of the agent to be encapsulated out of the first fluid occurs.It has been found that miscible fluids can require more care thanimmiscible systems in regulating diffusion times. In some cases, it maybe beneficial to increase the viscosity of the first fluid, therebyslowing diffusion of the agent to be encapsulated out of the inner core.In some cases, it may be beneficial to reduce the time needed forformation of the encapsulating shell. For encapsulating shells formedfrom encapsulating agents with a hydrophobic component, theencapsulating shell may be formed more quickly if the second liquidinitially contains some water. In an embodiment, the upper limit on thewater content of the second layer is that at which the second liquid nolonger dissolves the lipid or other encapsulating agent with ahydrophobic component. In this instance, the lipids would coalesce intoa bilayer before contacting the “core” fluid and would merely be insuspension (as opposed to solution). Thus, encapsulation may not occurbecause self-assembly happened prematurely. The encapsulating shell mayalso be formed more quickly if evaporation of the second liquid isenhanced. Evaporation of the second liquid can be enhanced throughdrying or through use of vacuum.

The first fluid comprises an agent to be encapsulated. In an embodiment,the first liquid is a carrier for the agent to be encapsulated and iscapable of dissolving or suspending the agent to be encapsulated. In anembodiment, the first liquid is aqueous. The agent to be encapsulatedcan be a variety of materials. In an embodiment, the agent to beencapsulated is selected from the group consisting of polynucleotides,proteins, and bioactive agents. As used herein, a polynucleotide is atleast 10 nucleotides in length. The polynucleotide may be found innature, synthetic, or any modified forms thereof. The polynucleotide maybe single- or double-stranded. The polynucleotide can be, but is notlimited to, DNA, RNA, chemical modifications thereof (e.g.,phosphorothioates, LNA), and polynucleotide complexes. Complexesincorporating polynucleotides include DNA condensates. In an embodiment,the protein is hemoglobin.

In an embodiment, the agent to be encapsulated is a bioactive agent. Asused herein a bioactive agent is a substance which may be administeredto any biological system, such as an organism, preferably a human oranimal host, and causes some biological reaction. Bioactive agentsinclude pharmaceutical substances, where the substance is administerednormally for a curative or therapeutic purpose. In an embodiment, thebioactive agent is a water-soluble pharmaceutical. The bioactive agentcan be, but is not limited to: adrenergic agent; adrenocortical steroid;adrenocortical suppressant; aldosterone antagonist; amino acid;anabolic; analeptic; analgesic; anesthetic; anorectic; anti-acne agent;anti-adrenergic; anti-allergic; anti-amebic; anti-anemic; anti-anginal;anti-arthritic; anti-asthmatic; anti-atherosclerotic; antibacterial;anticholinergic; anticoagulant; anticonvulsant; antidepressant;antidiabetic; antidiarrheal; antidiuretic; anti-emetic; anti-epileptic;antifibrinolytic; antifungal; antihemorrhagic; antihistamine;antihyperlipidemia; antihypertensive; antihypotensive; anti-infective;anti-inflammatory; antimicrobial; antimigraine; antimitotic;antimycotic, antinauseant, antineoplastic, antineutropenic,antiparasitic; antiproliferative; antipsychotic; antirheumatic;antiseborrheic; antisecretory; antispasmodic; antithrombotic;anti-ulcerative; antiviral; appetite suppressant; blood glucoseregulator; bone resorption inhibitor; bronchodilator; cardiovascularagent; cholinergic; depressant; diagnostic aid; diuretic; dopaminergicagent; estrogen receptor agonist; fibrinolytic; fluorescent agent; freeoxygen radical scavenger; gastrointestinal motility effector;glucocorticoid; hair growth stimulant; hemostatic; histamine H2 receptorantagonists; hormone; hypocholesterolemic; hypoglycemic; hypolipidemic;hypotensive; imaging agent; immunizing agent; immunomodulator;immunoregulator; immunostimulant; immunosuppressant; keratolytic; LHRHagonist; mood regulator; mucolytic; mydriatic; nasal decongestant;neuromuscular blocking agent; neuroprotective; NMDA antagonist;non-hormonal sterol derivative; plasminogen activator; plateletactivating factor antagonist; platelet aggregation inhibitor;psychotropic; radioactive agent; scabicide; sclerosing agent; sedative;sedative-hypnotic; selective adenosine Al antagonist; serotoninantagonist; serotonin inhibitor; serotonin receptor antagonist; steroid;thyroid hormone; thyroid inhibitor; thyromimetic; tranquilizer;amyotrophic lateral sclerosis agent; cerebral ischemia agent; Paget'sdisease agent; unstable angina agent; vasoconstrictor; vasodilator;wound healing agent; xanthine oxidase inhibitor.

Bioactive agents include immunological agents such as allergens (e.g.,cat dander, birch pollen, house dust, mite, grass pollen, etc.) andantigens from pathogens such as viruses, bacteria, fungi and parasites.These antigens may be in the form of whole inactivated organisms,peptides, proteins, glycoproteins, carbohydrates or combinationsthereof. Specific examples of pharmacological or immunological agentsthat fall within the above-mentioned categories and that have beenapproved for human use may be found in the published literature.

Experimental studies have investigated the effect of atomization on DNAstructural integrity, and clearly demonstrated that the proposedelectrostatic spray process does not degrade plasmid, cosmid, or linearforms of the macromolecule (Lentz et al., 2003). Electrostatic atomizersimpart minimal strain rates on the fluid (Table 1), and therefore, donot degrade the molecular structure through shear forces. No degradationis observed regardless of applied voltage (1 to 10 kV) except when thecharge density exceeds physical limits (as indicated by a coronadischarge).

In another embodiment, the agent to be encapsulated is a food componentor a cosmetic component. In an embodiment, the food component is a foodadditive.

The droplet size can be selected so that the agent to be encapsulatedfits within the droplet. Without wishing to be bound by any particulartheory, it is believed that so long as the molecule remains smaller thanthe interior cavity or inner core the recoil velocity imparted to theliquid filament between the droplet and liquid column during pinch offshould be sufficient to force any strands of the agent to beencapsulated extending into the filament back into the droplet interior.In an embodiment, the agent to be encapsulated is DNA. It may bedesirable to complex plasmid DNA to cationic agents (e.g. PEI,protamine) to condense the DNA to a smaller size. Complexation of largeplasmids (approximately 300 nm in diameter) is known to condense DNA tosizes well below 100 nm. In the practice of the invention, thesecationic agents would be neutralized by the DNA and fully encapsulated,thereby avoiding possible adverse interactions with blood components.

The second fluid comprises an encapsulating agent. In an embodiment, thesecond liquid is a carrier for the agent to be encapsulated and iscapable of dissolving or suspending the encapsulating agent. The secondliquid is miscible with the first liquid. The encapsulating agent can beselected from the group consisting of lipids, polymers, and polymerprecursors. Useful polymers include synthetic and natural polymers. Inan embodiment, the encapsulating agent can be selected from the groupconsisting of lipids, and polymers. Natural polymers includepolysaccharides and proteins. The concentration of the encapsulatingagent in the second liquid is sufficient to form a complete shell aroundthe agent to be encapsulated.

Useful lipid encapsulating agents are amphipathic lipids capable offorming a lipid bilayer. Useful lipid encapsulating agents includephospholipids, glycolipids, lipoproteins, sulfolipids, and mixturesthereof. As used herein, phospholipids include both glycerophospholipidsand sphingosyl phosphatides. Phospholipids useful in the presentinvention include phosphatidic acids, choline glycerophospholipids,serine glycerophospholipids, ethanolamine glycerophospholipids,phosphoinositides, sphigomyelin, and mixtures thereof. Also useful forthe present invention are phospholipid derivatives includingphospholipids grafted to polymers. The lipid bilayer can alsoincorporate sterols, proteins, glycoproteins or other agents that areeither temperature- or pH- sensitive and facilitate targeting, uptake orefficacy. The lipid bilayer can be neutral or negatively-charged.

Suitable solvents for lipid encapsulating agents include organicsolvents such as alcohols, acetone, DMSO, PEG, and glycerols, mixturesof organic solvents and mixtures of organic solvents with water. In anembodiment, the solvent is selected from alcohols, alcohol mixtures andalcohol-water mixtures. Suitable alcohols include, but are not limitedto, ethanol, methanol and isopropanol.

In an embodiment, the encapsulating shell is formed by precipitation ofthe polymer upon at least partial removal of the second liquid. In thisembodiment, the polymer is selected so that it is not substantiallysoluble in the first liquid. In an embodiment, the first liquid isaqueous and the polymer has a hydrophobic component. Usefulencapsulating agents for this embodiment include both synthetic andnatural polymers.

The polymer may be any suitable microencapsulation material including,but not limited to, nonbioerodable and nonbioerodable polymers. Suchpolymers have been described in great detail in the prior art. Theyinclude, but are not limited to: polyamides, polycarbonates,polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkyleneterepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters,polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes and copolymers thereof, alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses,polymers of acrylic and methacrylic esters, methyl cellulose, ethylcellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose,hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate,cellulose acetate butyrate, cellulose acetate phthalate, carboxylethylcellulose, cellulose triacetate, cellulose sulphate sodium salt,poly(methyl methacrylate), poly(ethylmethacrylate),poly(butylmethacrylate), poly(isobutylmethacrylate),poly(hexlmethacrylate), poly(isodecylmethacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecylacrylate), polyethylene, polypropylene poly(ethylene glycol),poly(ethylene oxide), poly(ethylene terephthalate), poly(vinylalcohols), poly(vinyl acetate, poly vinyl chloridepolystyrene,polyvinylpyrrolidone and polyethylenimine. In an embodiment,the polymer is polyethylenimine (PEI).

Examples of preferred biodegradable and bioerodable polymers includesynthetic polymers such as polymers of lactic acid and glycolic acid,polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid),poly(valeric acid), poly(caprolactone), poly(hydroxybutyrate),poly(lactide-co-glycolide) and poly(lactide-co-caprolactone), andnatural polymers such as algninate and other polysaccharides includingdextran and cellulose, collagen, albumin and other proteins andpeptides, zein and other prolamines and hydrophobic proteins. Alsouseful are chemical derivatives of the above polymers (substitutions,additions of chemical groups, for example, alkyl, alkylene,hydroxylations, oxidations, and other modifications routinely made bythose skilled in the art). In general, these materials degrade either byenzymatic hydrolysis or exposure to water in vivo, by surface or bulkerosion. The foregoing materials may be used alone, as physical mixtures(blends), or as co-polymers. Preferred biodegradable polymers are polylactides, poly lactide co-glycolides, chitosan, polylysines,polyarginines and biodegradable PEI derivatives (for example, Forrest,M. K. et al. “A Degradable Polyethylenimine Derivative with Low Toxicityfor Highly Efficient Gene Delivery”, Bioconjugate Chem., 2003, 14,934-940).

In this embodiment, polymer and protein solvents will typically be acommon organic solvent such as a halogenated aliphatic hydrocarbon suchas methylene chloride, chloroform and the like; an alcohol; an aromatichydrocarbon such as toluene; a halogenated aromatic hydrocarbon; anether such as methyl t-butyl; a cyclic ether such as tetrahydrofuran;ethyl acetate; diethylcarbonate; acetone; or cyclohexane. Suitablesolvents include low molecular weight polyethyleneglycol, acetone,dimethylsulfoxide, and glycerol. Suitable solvents for polysaccharideencapsulating agents include alcohols, low molecular weightpolyethyleneglycol, acetone, dimethylsulfoxide, and glycerol.

In another embodiment, the polymer is an amphiphilic molecule which selfassembles into a vesicle structure. Polymers of this type includediblock copolymers (Discher, B. M. et al. (1999) “Polymersomes: ToughVesicles Made from Diblock Copolymers”, Science, 284: 1143-1146),carbohydrate-based polymers (Uchegbu, I. F. et al., (1998) “Polymericchitosan based vesicles for drug delivery. J. Pharm. Pharmacol., 50,453-8) and amino acid based polymers (Uchegbu, I. F. et al., (1998)“Polymeric vesicles from amino acid homopolymers”, Proc. Intl. Symp.Control. Rel. Bioact. Mater. 25, 186-7).

In another embodiment, the encapsulating shell is formed bycross-linking of a polymer precursor encapsulating agent. Thepolymerization process can be initiated by photo-irradiation, thermally,or chemically. A polymer precursor means a molecule or portion thereofwhich can be polymerized to form a polymer or copolymer. Polymerprecursors include any substance that contains an unsaturated moiety orother functionality that can be used in chain polymerization, or othermoiety that may be polymerized in other ways. Such precursors includemonomers and oligomers. Preferred precursors include those that arecapable of being polymerized by photo radiation. Some examples ofprecursors that are useful in the invention include ethylene oxides (forexample, PEO), ethylene glycols (for example, PEG), vinyl alcohols (forexample, PVA), vinyl pyrrolidones (for example, PVP), ethyloxazolines(for example, PEOX), amino acids, saccharides, proteins, anhydrides,vinyl ethers, amides, carbonates, phenylene oxides (for example, PPO),acetals, sulfones, phenylene sulfides (for example, PPS), esters,fluoropolymers, imides, amide-imides, etherimides, ionomers,aryletherketones, olefins, styrenes, vinyl chlorides, ethylenes,acrylates, methacrylates, amines, phenols, acids, nitriles, acrylamides,maleates, benzenes, epoxies, cinnamates, azoles, silanes, chlorides,epoxides, lactones and amides. In an embodiment, the polymer precursoris a hydro acid, L-lactic acid, D,L-lactic acid, glycolic acid,copolymers thereof or a polyanhydride. In another embodiment, thepolymer precursor is an ethylenimine.

In this embodiment, the second fluid will typically further comprise aninitiator. If a polymer precursor that polymerizes photochemically isused (photosensitive polymer precursor), a separate photoinitator doesnot need to be used. Examples of photosensitive polymer precursorsinclude tetramercaptopropionate and 3,6,9,12-tetraoxatetradeca-1,13-diene. Depending upon the cross-linking method, the first fluid mayfurther comprise an agent to protect the agent to be encapsulated duringthe cross-linking process.

Photoinitiators that are useful in the invention include those that canbe activated with light and initiate polymerization of the polymerprecursor. Preferred initiators include azobisisobutyronitrile,peroxides, phenones, ethers, quinones, acids, formates. Cationicinitiators are also useful in the invention. Preferred cationicinitiators include aryldiazonium, diaryliodonium, and triarylsulfoniumsalts. Most preferred initiators include Rose Bengal (Aldrich), Darocur2959 (2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone, D2959,Ciba-Geigy), Irgacure 651 (2,2-dimethoxy-2-phenylacetophenone, I651,DMPA, Ciba-Geigy), Irgacure 184 (1-hydroxycyclohexyl phenyl ketone,I184, Ciba-Geigy), Irgacure 907(2-methyl-1-[4-(methylthio)phenyl]-2-(4-morpholinyl)-1-propanone, I907,Ciba-Geigy), Camphorquinone (CQ, Aldrich), isopropyl thioxanthone(quantacure ITX, Great Lakes Fine Chemicals LTD., Cheshire, England). CQis typically used in conjunction with an amine such as ethyl4-N,N-dimethylaminobenzoate (4EDMAB, Aldrich) or triethanolamine (TEA,Aldrich) to initiate polymerization.

The wavelengths and power of light useful to initiate polymerizationdepends on the initiator used or the wavelength (or wavelengths) whichwill activate the photosensitive precursor. A combination ofphotosensitive precursor(s) and photoinitiator(s) may be used. When RoseBengal is used as the initiator, a visible light source is preferablyused. Light used in the invention includes any wavelength and powercapable of initiating polymerization. Preferred wavelengths of lightinclude ultraviolet or visible. Any suitable source may be used,including laser sources. The source may be broadband or narrowband, or acombination.

An additional complete or partial layer of a third fluid can be formedon the outside of the second fluid layer. In an embodiment, this thirdfluid is aqueous. In an embodiment, the third fluid comprises atargeting and/or steric stabilizing agent which incorporates into thelipid bilayer shell. As used herein, a targeting agent facilitatescellular localization and/or uptake. Suitable targeting agents areligands which will bind specifically to receptor sites on the surface ofa target cell. Ligand-mediated targeting of liposomes is known to theart (Jones, M. N. and Chapman, D., (1995), Micelles, Monolayers andBiomembranes, Wiley-Liss, pp126-134). Suitable targeting agents includepeptides, lectins, aptamers, glycolipids, glycoproteins, viral spikeglycoproteins, antibodies, lipopolysaccharides, polysaccharides, andpolyarginine. In an embodiment, the targeting agent is selected from thegroup consisting of peptides, antibodies, lectins, sugars, and aptamers.As used herein, a steric stabilizing agent modifies stability inphysiological conditions. In an embodiment, the steric stabilizing agentis selected from the group consisting of polyethyleneglycol (PEG),glycolipids, gangliosides, polyanions, polycations, hyaluronic acid,starch, dextrans, and sulfur-containing compounds.

As is known to the art, targeting agents can be conjugated to a moietythat will allow incorporation or attachment to the “shell.” For example,compounds with a hydrophobic moiety can insert into lipid bilayers. Ofcourse, these targeting agents can also be attached to hydrophilicgroups on the surface.

These drop-in-drop products 30 can be collected for further processing.For example, the products 30 can be collected in any fluid that provideintact deposition of the compound droplet. Specifically the mass of thedroplet must be sufficient to over come the surface tension at theinterface of the collection pool. Suitable collection methods includesolidification of the particle prior to impact by liquid nitrogen, anddeposition in aqueous solutions. In an embodiment, for example, theapplication of a vacuum around the pool will increase the blowingvelocity at the surface and reduce surface tension to near zero. In anembodiment, surfactants and other surface active agents may be added tothe collection pool to expand the applicable fluid range. The collectionbath may be grounded. After formation, the charge on the droplets may bereduced or eliminated by methods known to the art, including coronadischarge and radiation. The droplets may also be dried beforecollection.

The cost-efficient production of vectors using the proposedelectrostatic co-extrusion technique can be limited by the low flowrates used in product formation. Two techniques have been used toincrease liquid flow rates; (1) air assist and (2) capillary arrays. Airassist methods have only yielded increases in flow rates ofapproximately 8-fold (Regele et al., 2002). In contrast, capillaryarrays have an unlimited potential for scale up. Microlaminatetechnology could be employed to create nozzle arrays in a small space.However, it is important to realize that charged liquid streams flowingin parallel will form individual magnetic fields that may interfere withthe particle formation process. Previous work by Regele et al. (2002)suggests that careful distribution of the nozzle arrays should allow usto overcome these weak magnetic interactions. If the increases in arraydimensions result in large magnetic fields that are difficult toovercome with voltage potential, air assist may be required tofacilitate the breakup of the liquid column, thereby reducing themagnitude of the applied voltage. In an embodiment, the methods andapparatus of the invention employ gas flow to assist in atomization ofthe fluids. In this embodiment, gas can be flowed external to the fluidsto assist in “focussing” the fluids.

In an embodiment, the invention comprises an apparatus forelectrostatically atomizing a first and a second fluid, the apparatuscomprising:

-   -   a. a multiplicity of flow channel assemblies, each flow channel        assembly comprising a first fluid flow channel and a second        fluid flow channel; the first and second fluid flow channels of        each flow channel assembly being substantially coaxial;    -   b. a multiplicity of peripheral conductors;    -   C. a ground electrode;    -   d. a source of electrical potential electrically connected        between the ground electrode and the flow channel assemblies and        conductors;    -   e. at least one first liquid pump in fluid communication with        the first fluid flow channels of each flow channel assembly; and    -   f. at least one second liquid pump in fluid communication with        the second fluid flow channels of each flow channel assembly.

A flow channel assembly comprises the coaxial flow channels for thefluids which are electrostatically atomized during operation of theapparatus. The flow channel assembly may also be referred to as anozzle. The flow channel assembly is electrically connected to thesource of electric potential so that charge can be transferred to atleast one of the fluids which flow through the flow channel assemblyduring operation. For example, a flow channel may be at least partiallyelectrically conducting and the electrically conducting portion of theflow channel connected to the source of electrical potential. The flowchannel may also contain an electrode which is connected to the sourceof electrical potential.

The flow channel assemblies are surrounded or bordered by a multiplicityof peripheral conductors, as is schematically shown in FIG. 6. In FIG.6, the flow channel assemblies 60 are dark and the peripheral conductors70 are light. A peripheral conductor can be a dummy flow channel or flowchannel assembly which does not conduct fluid during operation of theapparatus (i.e. the flow channel or flow channel assembly is not influid communication with one or more liquid pumps). When the flowchannel assemblies are arranged so that some of the assemblies areperipheral to others (as is FIG. 6), the peripheral conductors help tomake the electric fields experienced by the peripheral flow channelassemblies more similar to those experienced by the more central flowchannel assemblies.

The flow channel assemblies and peripheral conductors can be made usingmicrofluidic fabrication techniques as are known to the art.Microfluidic fabrication techniques include standard photolithographicprocedures to form structures. Precision injection molded plastics mayalso be used for fabrication. The feature sizes and geometries areproducible by such methods as LIGA, thermoplastic micropattem transfer,resin based microcasting, micromolding in capillaries (MIMIC), wetisotropic and anisotropic etching, laser assisted chemical etching(LACE), and reactive ion etching (RIE), or other techniques known withinthe art of microfabrication.

The source of electrical potential provides a direct current potentialdifference between the ground electrode and the flow channel assembliesand the ground electrode and the peripheral conductors. In anembodiment, the apparatus uses more than one source of electricalpotential. For example, it may be desirable that a different source ofelectrical potential be used between the ground electrode and theperipheral conductors than the ground electrode and the flow channelassemblies. In an embodiment, the source of electrical potential is ahigh voltage power supply. As the spacing between the flow channelassemblies decreases, the required potential difference for electrosprayfrom the flow channel assemblies is expected to increase.

The liquid pumps supply liquid to the flow channels of the flow channelassemblies. The pumps may be of any kind known to the art which canprovide controlled flow at relatively low flow rates. In an embodiment,the liquid pump is a syringe pump, peristaltic pump, gravity feedreservoir, pressure driven reservoir, or any system providingcontrollable flow rates with only minor oscillatory behavior.

Although the description above contains much specificity, these shouldnot be construed as limiting the scope of the invention but as merelyproviding illustrations of some of the presently preferred embodimentsof the invention. Thus the scope of the invention should be determinedby the appended claims and the legal equivalents, rather than by theexamples given. All references cited herein are hereby incorporated byreference to the extent not inconsistent with the disclosure herein.

EXAMPLES Example 1 Formation of Aqueous Droplets Encapsulated by a LipidBilaver

Methylene blue (saturated) dissolved in water was flowed through theinner flow channel and egg phosphatidylcholine (0.62 mg/ml) dissolved inethanol was flowed through the outer channel of an electrostaticatomization apparatus. The inner flow channel was an inner quartzcapillary with an inner diameter of about 0.25 mm and an outer diameterof about 0.321 mm. The outer flow channel was a stainless capillary withan inner diameter of approximately 0.564 mm and an outer diameter ofapproximately 1.069 mm. The inner and outer flow channel weresubstantially coaxial. Flow rates for both solutions were about 0.3ml/min and achieved using two syringe pumps (Harvard Apparatus, InfusionPump, model 940). The applied voltage was approximately 5-6 kV and theorifice to ground distance was approximately 10 mm. The voltage wasapplied to only the inner capillary by connecting it to a metal tube.

FIG. 5 shows a water droplet (clear interior) encapsulated in a lipidshell (dark wall) formed in this manner. The sample was collected inliquid nitrogen and then transferred to water to thaw. Images were takenafter the vesicles had reached room temperature. The lipid layer isexaggerated in the image due to light diffraction. The droplet sizes inFIG. 5 are approximately 50 microns.

Example 2 Formation of Compound Droplets with an Aqueous Core and TwoOuter Fluid Lavers

Compound droplets were formed with a central region of methylene bluedissolved in water, an isopropanol layer surrounding the aqueous core,and a water layer surrounding the isopropanol layer. The electrostaticatomization apparatus was as described in Example 1, with the additionof an outermost flow channel. The outermost flow channel was ofstainless steel piping with an inner diameter of approximately 2 mm andan outer diameter of approximately 3 mm. TABLE 1 Flow rate of Flow rateof isopropanol in water in annular Flow rate of annular space spacebetween water in inner between inner middle and outer tube and middletube tube A 0.136 mL/min 0.136 mL/min 0.388 mL/min B  0.51 mL/min  0.34mL/min  0.51 mL/min C 0.103 mL/min 0.136 mL/min 0.206 mL/min

The applied voltage difference was approximately 5-6 kV. Flow rates wereas shown in Table 1.

Five Nozzle Array

FIG. 6 is a schematic of staggered nozzle array. Filled circlesrepresent the co-axial capillaries 60 producing the compound droplets.Empty circles represent false charged capillaries 70 used to establish auniform electric field. The five active nozzles are arranged in astaggered formation that minimizes the total space while maximizing thecenter to center distance between charged capillary tubes (FIG. 6). Inthis configuration the center capillary experiences an electric fieldsubstantially different than those on the perimeter; therefore, fielduniformity is increased using dummy tubes. The dummy tubes consist ofnon-flowing, charged capillaries at positions consistent with the nextlayer in the array. This square configuration also provides an easygeometry to replicate into larger nozzle arrays (e.g., 100 by 100nozzles). When the geometry is replicated, the pattern of five activenozzles is replicated and the resulting array of active nozzles issurrounded by dummy nozzles around the periphery.

The configuration shown in FIG. 6 has been constructed and producesTaylor cones of an aqueous solution surrounded by an alcohol.

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1. A method for making an encapsulated droplet comprising an agent to be encapsulated, the method comprising the steps of: a. forming a compound droplet through electrostatic atomization of a first and a second liquid, the compound droplet comprising a droplet of the first liquid surrounded by a layer of the second liquid, wherein the first liquid comprises an agent to be encapsulated, the second liquid comprises an encapsulating agent and is miscible with the first liquid and at least one of the first or second liquid is electrically conductive; and b. forming an encapsulating shell from the encapsulating agent, thereby forming an encapsulated droplet comprising an agent to be encapsulated.
 2. The method of claim 1, wherein the compound droplet is formed by simultaneously flowing the first and the second liquid through substantially coaxial flow channels, and applying electrical charge to at least one of the first or second fluids so that the electrical charge is applied to an electrically conducting fluid.
 3. The method of claim 1, wherein both the first and the second fluid are electrically conducting and electrical charge is applied to both of the fluids.
 4. The method of claim 1 wherein the agent to be encapsulated is a bioactive agent.
 5. The method of claim 1, wherein the agent to be encapsulated is selected from the group consisting of pharmaceuticals, polynuecleotides, and proteins.
 6. The method of claim 5, wherein the agent to be encapsulated is a polynucleotide selected from the group consisting of DNA, RNA, chemically modified polynucleotides and complexes containing polynucleotides.
 7. The method of claim 1, wherein the agent to be encapsulated is a food component or cosmetic component.
 8. The method of claim 1, wherein the encapsulating agent is selected from the group consisting of lipids, polymers and polymer precursors.
 9. The method of claim 1, wherein the first fluid is aqueous.
 10. The method of claim 9, wherein the second liquid is an alcohol.
 11. A method for making an encapsulated droplet comprising an agent to be encapsulated, the method comprising the steps of: a. forming a compound droplet through electrostatic atomization of a first, a second, and a third liquid, the compound droplet comprising a droplet of the first liquid surrounded by a layer of the second liquid with the third liquid forming a complete or partial layer around the second liquid, wherein the first liquid comprises an agent to be encapsulated, the second liquid comprises an encapsulating agent and is miscible with the first liquid and the third liquid comprises a targeting or steric stabilizing agent and at least one of the liquids is electrically conductive; and b. forming an encapsulating shell from the encapsulating agent, thereby forming an encapsulated droplet comprising a first agent to be encapsulated.
 12. The method of claim 11 wherein the compound droplet is formed by simultaneously flowing the three liquids through substantially coaxial flow channels, and applying electrical charge to at least one of the three liquids so that the electrical charge is applied to an electrically conductive fluid.
 13. The method of claim 12 wherein electrical charge is applied to the first and the second fluids.
 14. The method of claim 11 wherein the first fluid is aqueous.
 15. The method of claim 11 wherein the third liquid is miscible with the second liquid.
 16. The method of claim 11 wherein the third liquid is aqueous.
 17. The method of claim 11 wherein the targeting agent is selected from the group consisting of: peptides, antibodies, lectins, sugars, and aptamers.
 18. The method of claim 11 wherein the steric stabilizing agent is selected from the group consisting of polyethyleneglycol, glycolipids, gangliosides, polyanions, polycations, hyaluronic acid, starch, dextrans, and sulfur-containing compounds.
 19. An apparatus for electrostatically atomizing a first and a second fluid, the apparatus comprising: a. a multiplicity of flow channel assemblies, each flow channel assembly comprising a first fluid flow channel and a second fluid flow channel; the first and second fluid flow channels of each flow channel assembly being substantially coaxial; b. a multiplicity of peripheral conductors; c. a ground electrode; d. a source of electrical potential electrically connected between the ground electrode and the flow channel assemblies and the peripheral conductors; e. at least one first liquid pump in fluid communication with the first fluid flow channels of each flow channel assembly; and f. at least one second liquid pump in fluid communication with the second fluid flow channels of each flow channel assembly.
 20. The apparatus of claim 19, wherein five flow channel assemblies are arranged to form a pattern such that one assembly is in the center and the other four assemblies are substantially equidistant from the central assembly and are substantially equidistant from each other.
 21. The apparatus of claim 20, wherein the pattern of five flow channel assemblies is repeated. 